XBB.1.5 spike protein COVID-19 vaccine induces broadly neutralizing and cellular immune responses against EG.5.1 and emerging XBB variants

Monovalent SARS-CoV-2 Prototype (Wuhan-Hu-1) and bivalent (Prototype + BA.4/5) COVID-19 vaccines have demonstrated a waning of vaccine-mediated immunity highlighted by lower neutralizing antibody responses against SARS-CoV-2 Omicron XBB sub-variants. The reduction of humoral immunity due to the rapid evolution of SARS-CoV-2 has signaled the need for an update to vaccine composition. A strain change for all authorized/approved vaccines to a monovalent composition with Omicron subvariant XBB.1.5 has been supported by the WHO, EMA, and FDA. Here, we demonstrate that immunization with a monovalent recombinant spike protein COVID-19 vaccine (Novavax, Inc.) based on the subvariant XBB.1.5 induces neutralizing antibodies against XBB.1.5, XBB.1.16, XBB.2.3, EG.5.1, and XBB.1.16.6 subvariants, promotes higher pseudovirus neutralizing antibody titers than bivalent (Prototype + XBB.1.5) vaccine, induces SARS-CoV-2 spike-specific Th1-biased CD4 + T-cell responses against XBB subvariants, and robustly boosts antibody responses in mice and nonhuman primates primed with a variety of monovalent and bivalent vaccines. Together, these data support updating the Novavax vaccine to a monovalent XBB.1.5 formulation for the 2023–2024 COVID-19 vaccination campaign.


XBB lineage booster immunization in mice and nonhuman primates
We also investigated immune responses in mice primed with bivalent rS (Prototype rS + BA.5 rS) followed by a booster dose of monovalent XBB.1.5 or XBB.1.16.Groups of mice (n = 10 per group) were inoculated intramuscularly with a primary immunization series of a bivalent (Prototype rS + BA.5 rS) vaccine on days 0 and 14, followed by a single booster dose with XBB.1.5rS or XBB.1.16rS on day 47.Sera were collected at day 21 (1 week after the second dose) and day 61 (2 weeks after booster dose).XBB.1.5 and XBB.1.16induced a > 35-fold increase in pseudovirus neutralizing antibodies against XBB.1.5 and XBB.1.16compared to the titers after the primary series (Fig. 2a).For all variant pseudoviruses examined, including forward drifted variants FL.1.5.1 and EG.5.1, neutralizing titers were not statistically significantly different after a booster with either XBB.1.5rS or XBB.1.16rS, with the exception of Omicron BA.5, for which the booster with XBB.1.5rS resulted in higher antibody titers (Fig. 2a).A booster dose with XBB.1.5rS resulted in detectable antibody titers against BA.2.86, which were undetectable after the primary series with bivalent Prototype rS + BA.5 rS.Pseudovirus neutralizing antibody titers in mice were further analyzed by antigenic cartography, a method for visualizing antigenic diversity.Priming with two doses of bivalent vaccine (Prototype + BA.5) resulted in greater than 30-fold differences in neutralizing responses between Prototype to both XBB.1.5 and XBB.1.16shown by antigenic cartography (Fig. 2b).This large antigenic distance was expected as the neutralizing epitopes of Prototype and BA.5 rS are mainly absent on XBB sub-variants.Antigenic distances with a fold-difference less than twofold are considered to be matched responses.Boosting primed mice with XBB.1.5vaccine induced a matched response to XBB.1.16,with an antigenic distance of 0.691 (Fig. 2b).Similarly, an XBB.1.16boost induced a matched response to XBB.1.5 with a fold-difference of 0.750 (Fig. 2b).

Discussion
Neutralizing antibodies inhibit viral infections by binding viral surface components that participate in host-cell fusion and entry.The generation of neutralizing antibodies specific for the envelope embedded SARS-CoV-2 spike glycoprotein following infection or vaccination is a crucial part of a functional SARS-CoV-2 immune response.Neutralizing antibodies can also be effective when used as prophylactic or therapeutic treatments against COVID-19  [21][22][23][24][25][26][27] , the robust pseudovirus neutralizing antibody titers generated after immunization with XBB.1.5rS vaccine observed in these preclinical studies will likely translate to favorable responses in the clinic against contemporary SARS-CoV-2 variants of concern.The cell-mediated immunogenicity of the monovalent XBB.1.5vaccine was further evaluated by measuring cell-mediated immune responses, which showed the presence of a polyfunctional Th1-biased CD4+ response against XBB sub-variants in mice and NHPs.

Animal ethics statement
The reporting in this manuscript follows the recommendations in the ARRIVE guidelines.
The mouse studies were conducted at Noble Life Sciences (Sykesville, MD
For the booster study, female BALB/c mice (N = 10 per group, 200 mice total) were immunized by intramuscular (IM) injection with two 1 µg doses spaced 14 days apart (study day 0 and 14) of Prototype (control) or Prototype + Omicron BA.5 (0.5 µg each) with 5 μg Matrix-M adjuvant.A booster (3 rd dose) of 1 µg Omicron XBB.1.5 or XBB.1.16with 5 μg Matrix-M adjuvant was administered on Day 47 (approximately 1 month post 2nd dose).Sera and spleen were collected 2 weeks after the booster dose on day 61 to evaluate antibody and cellular responses.
For all mouse studies, animals were randomly assigned to groups as they were removed from shipping cages.

Nonhuman primate study design
Female and male rhesus macaques (Macaca mulatta, N = 5 per group; N = 15 total), 3-11 years old and weighing 3-9 kg at study initiation, were obtained from a SNPRC specific pathogen free (SPF) colony and/or Envigo (Alice, TX).Animals were randomly assigned to groups based on similar age and sex distribution across the groups.NHPs were immunized by intramuscular injection (0.5 mL) with the human dose level: 5 µg NVX SARS-CoV-2 Prototype (control) or Omicron BA.5 variant rS vaccines adjuvanted with 50 µg Matrix-M administered as monovalent, or bivalent prime/boost on days 0 and 21 (primary series).A booster consisting of Omicron XBB.1.5rS with 50 µg Matrix-M was administered at week 35 (Day 246).Sera were collected prior to the boost on Day 210, as well as 2 weeks post boost on Day 260.Peripheral blood mononuclear cells (PBMCs) were also collected on Day 260.

Pseudovirus neutralization
SARS-CoV-2 pseudoviruses were generated using a lentivirus platform adapted from Crawford, 2020 32 .Briefly, backbone and helper plasmids, including Wuhan-Hu-1 spike, were obtained from BEI Resources.Additional variants were synthesized in pcDNA3.1 (GenScript) using the appropriate spike protein sequence from the EPICoV database.All spike protein sequences included a deletion of the cytoplasmic tail.HEK293T cells were seeded one day prior to transfection, incubated at 37 °C overnight, and transfected when the cellular monolayer was 60-75% confluent.The transfection uses a cationic-lipid delivery system such as Lipofectamine 3000 (Thermo Fisher) or JetPrime Optimus (Polyplus) with a set of plasmids encoding: a lentiviral backbone, a dual reporter plasmid expressing both luciferase and Zs green, a plasmid expressing SARS-CoV-2 spike (such as Wuhan-Hu-1, Omicron BA.5, and BQ.1.1)and a plasmid expressing other HIV proteins for pseudovirion formation.Then, 48 h following transfection, supernatants were collected, centrifuged, and filtered through a 0.45 µm filter to obtain a pseudovirus stock.Commercial pseudovirus for Omicron XBB.1.5,XBB.1.16,XBB.2.3, and EG.5.1 were obtained from eEnzyme and incorporated only a luciferase reporter gene for detection of pseudoviral entry.Aliquots of pseudovirus stock were stored at − 80 °C.All work with pseudovirus was performed in a Biosafety Level 2 laboratory and approved by our Institutional Biosafety Committee.Each newly produced lot or new shipment of pseudovirus, if commercially obtained, was titered under assay conditions to determine the working dilution to target an RLU of 100,000 prior to testing serum.The pseudovirus neutralization assay was then performed using a HEK293T cell line stably expressing hACE2 (HEK293T/ ACE2 obtained from Creative Biogene).Serum samples were heat-inactivated by placing in a 56 °C water bath for 30 min, followed by cooling to 4 °C immediately.Serum samples were serially diluted three-fold in reduced serum Opti-MEM starting at a 1:20 or 1:50 dilution in a 96-well tissue culture plate.Fifty microliters of SARS-CoV-2 Pseudovirus stock (corresponding to 100,000 RLU, range from 50,000-250,000) was then added to each well, followed by incubation at 37 °C for one hour.Then, 2.0 × 10 4 HEK293T/hACE2 cells in 100 µL of HEK293T cell culture medium (DMEM without phenol red + 5% FBS + 1% Penicillin + streptomycin + glutamine) containing 1.25 µg/mL puromycin were added to the wells, followed by incubation for 72 h at 37 °C.After incubation, 50 µL BrightGlo Luciferase Substrate (Promega) was added to each well.Plates were incubated for 5 min at room temperature without ambient light.Viral entry into the cells was determined by measuring the luminescence with a SpectraMax iD3 microplate reader.Pseudovirus neutralizing antibody titer of the serum was determined through the absence or reduction of luminescence in a well.Data were analyzed and neutralization curves were generated in GraphPad Prism for each sample; 50% pseudovirus neutralization titers (pVN 50 ) and 50% inhibition dilution (ID50) were calculated using 4-parameter curve fitting.No-serum wells were present on each plate along with at least one positive and negative monoclonal antibody for each pseudovirus tested.
A similar pseudovirus neutralization assay, validated for testing human samples (for Ancestral, Omicron BA.5 and XBB.1.5strains), was utilized as fit-for-purpose for testing NHP samples.This method is similar to the method used for the mouse studies except the input virus targeted an RLU of 50,000 (range 10,000-300,000) per well and diluted in infection medium containing Dulbecco's Minimal Essential Medium (DMEM) and 2% heat inactivated fetal bovine serum (FBS), test serum and pseudovirus was incubated for 2-h; 10,000 cells/well were used for the assay.Serum dilution series started at 1:10, which was reported as 1:20 after addition of virus.Luciferase readout was performed from 15 min after addition of luciferase reagent up to 60 min, followed by data calculation using Softmax 4-parameter curve fit.

Antigenic cartography
Pseudovirus neutralizing antibody titers in mouse sera were subjected to antigenic cartography analysis to visualize antigenic diversity (reviewed in 33 ).Antigenic cartography maps were constructed using Cartography software available at acmacs-web.antigenic-cartography.org.Pseudovirus neutralizing titers in sera collected on Day 21 (1 week after the two-dose primary immunization) and Day 61 (2 weeks after the booster) were input into the software to construct a SARS-CoV-2 antigenic map.SARS-CoV-2 antigens are depicted as circles and sera are indicated as small squares.Each grid square represents one antigenic unit representing a two-fold change in titers.The antigenic distances among Prototype, Omicron XBB.1.5,XBB.1.16,and XBB.2.3 variants were calculated after the primary series and after the booster dose.Antigenic Distances were converted to fold-differences.

Statistical analysis
Mann-Whitney U Tests (two-tailed) were used when determining statistical significance of differences between two groups, and Kruskal-Wallis Multiple Comparisons Test was used when comparing differences among three groups.GraphPad Prism 9.0 software (La Jolla, CA) was used to conduct statistical tests, calculate geometric mean titers (GMTs) and 95% confidence intervals (95% CIs), and plot data.

Figure 1 .
Figure 1.Humoral responses following variant-adapted two-dose primary series vaccination in mice.(a) Pseudovirus neutralization in mice sera collected one week following primary vaccination with two doses of monovalent XBB.1.5 or bivalent (Prototype + XBB.1.5).Sera were collected on Day 21 as indicated in the study design diagram.(b) Pseudovirus neutralization in mice sera collected one week following primary series vaccination with two doses of monovalent Prototype, XBB.1.5,or XBB.1.16.Sera were collected on Day 21 as indicated in the study design diagram.Note that all immunizations were administered with 5 µg Matrix-M adjuvant.Open circles represent individual data points, solid bars represent group geometric mean titers, error bars represent 95% confidence intervals, and the horizontal dashed line represents the assay limit of detection (LOD).Statistically significant differences are marked with asterisks: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.In Panel (b), asterisks represent differences that were significant between the Prototype rS group and the other two immunization groups.

Figure 2 .
Figure 2. Humoral responses following XBB.1.5booster in mice.(a) Pseudovirus neutralization titers were determined in mouse sera collected one week after a primary series with bivalent Prototype rS + BA.5 rS (left graph series), and following a boost with monovalent XBB.1.5(middle graph series) or XBB.1.16(right graph series) vaccines (sera collected two weeks after booster dose).Note that all immunizations were administered with 5 µg Matrix-M adjuvant.Open circles represent individual data points, solid bars represent group geometric mean titers, error bars represent 95% confidence intervals, and the horizontal dashed line represents the assay limit of detection (LOD).Statistically significant differences between the two booster groups are marked with asterisks: *P < 0.05.Pooled sera were analyzed for Day 21 data where indicated with an asterisk on the x-axis label.(b) Pseudovirus neutralizing titers presented in (a) were subjected to antigenic cartography analysis.Each small square corresponds to one animal and each grid square corresponds to one antigenic distance of twofold change in neutralization titer with fold differences given below each map.Smaller antigenic distance fold-change between two variants indicates higher cross-neutralizing antibody titers.

1 VolFigure 3 .
Figure 3. Humoral Responses Following XBB.1.5Booster in Rhesus Macaques.Pseudovirus neutralization titers in rhesus macaques boosted with XBB.1.5approximately 8 months after Prototype rS (top graph) or bivalent (Prototype + BA.5; bottom graph) priming regimens as shown in the study design diagram.Note that all immunizations were administered with 50 µg Matrix-M adjuvant.Sera were collected five weeks before the boost (Day 210) and two weeks after the booster dose (Day 260).Statistically significant differences in postboost titers between the two groups after are marked with asterisks above the group with the higher titers: *P < 0.05; **P < 0.01.Open circles represent individual data points, solid bars represent group geometric mean titers, error bars represent 95% confidence intervals, and the horizontal dashed line represents the assay limit of detection.

Figure 4 .
Figure 4. CD4 + T Cell Responses to an XBB.1.5Booster.(a) CD4 + T cell responses in mice primed with two doses of Prototype or bivalent (Prototype + BA.5) rS and boosted with XBB.1.5rS as outlined in Fig. 2. Splenocytes were collected two weeks after the booster dose.(b) CD4 + T cell responses in rhesus macaques primed with bivalent (Prototype + BA.5) rS and boosted with XBB.1.5rS.Peripheral blood mononuclear cells (PBMCs) were collected 2 weeks after the booster dose.Open circles represent individual animal data points, solid bars represent group geometric mean values with 95% CI error bars.
). Animals were maintained and treated according to Animal Welfare Act Regulations, the US Public Health Service Office of Laboratory Animal Welfare Policy on Humane Care and Use of Laboratory Animals, Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1996), and AAALACi accreditation.Mouse studies were approved by Noble Life Sciences IACUC.The study in rhesus macaques was conducted at Texas Biomedical Research Institute (San Antonio, TX).Animals were maintained at Texas Biomedical Research Institute for the entire in-life portion of the study and were treated according to Animal Welfare Act regulations and the Guide for the Care and Use of Laboratory Animals (2011).Rhesus macaque studies were approved by Texas Biomedical Research Institute IACUC.